The objective of this project is to elucidate the function of the dystrophin-glycoprotein complex in order to better understand the pathologies observed in Duchenne (DMD) and Becker (BMD) muscular dystrophies. Our studies thus far have substantially changed how dystrophin is thought to bind F-actin. They have demonstrated that dystrophin is necessary to mechanically couple costameric actin filaments to the sarcolemma and they have defined the actin-binding properties of utrophin. We now propose to address several unresolved aspects of dystrophin/utrophin interaction with known molecular partners and then characterize the structure and function of 4 therapeutically-important dystrophin mini- and micro-constructs expressed in the baculovirus system. We will assess the effect of DMD/BMD-causing point mutations on the structure, stability, and known ligand binding properties of dystrophin. The structures of dystrophin and utrophin in complex with F-actin will be further refined by electron microscopy and image reconstruction and the mechanical properties of each protein characterized by atomic force microscopy. Through immunofluorescence analysis of peeled sarcolemma and biochemical analysis of dystrophin-glycoprotein complex isolated from oc-dystrobrevin knockout mice, we will determine the role of a-dystrobrevin in anchoring dystrophin to the glycoprotein complex and in costamere stability. We will further investigate the contribution of a7 integrin in mechanically coupling actin filaments to the sarcolemma in normal muscle and to rescue coupling when over-expressed in the absence of dystrophin. Finally, we will amplify novel interacting proteins from muscle by dystrophin/utrophin affinity chromatography and identify novel partners by western blot analysis and MALDI-TOF mass spectrometry. ' Novel proteins will be further characterized by in vitro binding assays, co-immunoprecipitation and confocal microscopy analysis of control and dystrophic muscle. The proposed studies are expected to yield new insight into the function of the dystrophin-glycoprotein complex and costameres as well as muscular dystrophies where one or both are disrupted.